Anti-Angiogenic Cancer TherapiesAnti-Angiogenic

Cancer Therapies

Tangela S. FeemsterTuesday, March 27, 2007Dr. BuynakMedicinal Chemistry

Tangela S. FeemsterTuesday, March 27, 2007Dr. BuynakMedicinal Chemistry

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Definition of Angiogenic Therapy

Definition of Angiogenic Therapy

• A new form of cancer treatment using drugs called 'angiogenesis inhibitors' that specifically halt new blood vessel growth and starve a tumor by cutting off its blood supply.

• A substance in the body called Vascular Endothelial Growth Factor (VEGF) is responsible for the growth of new blood vessels. It promotes this growth by stimulating the endothelial cells, which form the walls of the vessels and transport nutrients and oxygen to the tissues.

• Anti-Angiogenic drugs prevent the VEGF from binding with the receptors on the surface of the endothelial cells.

• A new form of cancer treatment using drugs called 'angiogenesis inhibitors' that specifically halt new blood vessel growth and starve a tumor by cutting off its blood supply.

• A substance in the body called Vascular Endothelial Growth Factor (VEGF) is responsible for the growth of new blood vessels. It promotes this growth by stimulating the endothelial cells, which form the walls of the vessels and transport nutrients and oxygen to the tissues.

• Anti-Angiogenic drugs prevent the VEGF from binding with the receptors on the surface of the endothelial cells.

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Three Major Types of Anti-angiogenic Therapies for

Cancer

Three Major Types of Anti-angiogenic Therapies for

Cancer1. Drugs that stop new blood vessels

from sprouting (true angiogenesis inhibitors)

2. Drugs that attack a tumor's established blood supply (vascular targeting agents)

3. Drugs that attack both the cancer cells as well as blood vessel cells (the double-barreled approach).

1. Drugs that stop new blood vessels from sprouting (true angiogenesis inhibitors)

2. Drugs that attack a tumor's established blood supply (vascular targeting agents)

3. Drugs that attack both the cancer cells as well as blood vessel cells (the double-barreled approach).

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To date more than 300 angiogenesis inhibitor molecules have been discovered:Some angiogenesis inhibitors are naturally present in the human body because healthy tissues appear

to resist cancer growth by containing these anti-angiogenic compounds.

To date more than 300 angiogenesis inhibitor molecules have been discovered:Some angiogenesis inhibitors are naturally present in the human body because healthy tissues appear

to resist cancer growth by containing these anti-angiogenic compounds.

List of 32 Known Angiogenesis Inhibitors in the Body

Angiostatin (plasminogen fragment) Metalloproteinase inhibitors (TIMPs)

Anti-angiogenic antithrombin III (aaATIII) Pigment epithelial-derived factor (PEDF)

Canstatin Placental ribonuclease inhibitor

Cartilage-derived inhibitor (CDI) Plasminogen activator inhibitor

CD59 complement fragment Platelet factor-4 (PF4)

Endostatin (collagen XVIII fragment) Prolactin 16kD fragment

Fibronectin fragment Proliferin-related protein

Gro-beta Retinoids

Heparinases Tetrahydroco

Heparin hexasaccharide fragment rtisol-S

Human chorionic gonadotropin (hCG) Thrombospondin-1

Interferon alpha/beta/gamma Transforming growth factor-beta

Interferon inducible protein (IP-10) Tumistatin

Interleukin-12 (IL-12) Vasculostatin

Kringle 5 (plasminogen fragment) Vasostatin (calreticulin fragment)

2-Methoxyestradiol (2-d) Angioarrestin

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Angiogenesis InhibitorsAngiogenesis Inhibitors• Other angiogenesis inhibitors have been found in nature - in green tea, soy products, fungi, mushrooms, Chinese cabbage, tree bark, shark tissues, snake venom, red wine, and many other substances.

• Still other angiogenesis inhibitors have been manufactured synthetically in the laboratory.

• Some FDA-approved medicines have also been "re-discovered" to have anti-angiogenic properties.

• Other angiogenesis inhibitors have been found in nature - in green tea, soy products, fungi, mushrooms, Chinese cabbage, tree bark, shark tissues, snake venom, red wine, and many other substances.

• Still other angiogenesis inhibitors have been manufactured synthetically in the laboratory.

• Some FDA-approved medicines have also been "re-discovered" to have anti-angiogenic properties.

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Angiogenic InhibitorsAngiogenic Inhibitors

• Currently, a number of clinical trials in progress are combining anti-angiogenic therapy with cytotoxic chemotherapy or radiation, as a way to maximize the anti-tumor treatment in human cancer patients sponsored by biotechnology and pharmaceutical companies, medical centers and by the U.S. National Cancer Institute. These clinical trials are taking place in the United States, Canada, Australia, and throughout Europe.

• Currently, a number of clinical trials in progress are combining anti-angiogenic therapy with cytotoxic chemotherapy or radiation, as a way to maximize the anti-tumor treatment in human cancer patients sponsored by biotechnology and pharmaceutical companies, medical centers and by the U.S. National Cancer Institute. These clinical trials are taking place in the United States, Canada, Australia, and throughout Europe.

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Anti-Angiogenic Drugs in Clinical Trial for CancerAnti-Angiogenic Drugs in Clinical Trial for Cancer

A6Alpha5Beta1 Integrin AntibodyABT-510ActimidAngiocolAngiostatinAngiozymeAplidineAptosynATN-161Avastin (bevacizumab)AVE8062ABenefinBMS275291CarboxymidotriazoleCC4047CC7085CDC801Celebrex (Celecoxib)CEP-7055CGP-41251/PKC412Cilengitide

Combretastatin A4PCP-547, 632CP-564, 959DexrazoxaneDidemnin BDMXAAEMD 121974EndostatinFlavopiridolGBC-100Genistein Concentrated PolysaccharideGreen Tea ExtractInterleukin-12INGN 201Interferon alfaIressaLY317615Mab huJ591-DOTA-90 Yttrium (90Y)Medi-522Metaret (suramin)Metastat (Col-3)

NeovastatNM-3NPe6OctreotideOltiprazPaclitaxelPanzem (2ME2)PenicillaminePI-88PSKPTK787/ZK222584RevimidRo317453SqualamineSU11248SU6668TemptostatinTetrathiomolThalidomideUCN-01VEGF TrapZD6126ZD647

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Understanding AngiogenesisUnderstanding Angiogenesis

• Angiogenesis is defined as the growth of blood vessels and is an important natural process used by the body for reproduction and for healing injured tissues

• Blood vessels bring oxygen and nutrients via the circulation to nourish all tissues in the body

• The cells comprising blood vessels are called endothelial cells

• The endothelial cells of a blood vessel also produce molecules that support the growth of tissues

• Cancer cells take over the body's control of angiogenesis in order to recruit their own private blood supply

• Angiogenesis is defined as the growth of blood vessels and is an important natural process used by the body for reproduction and for healing injured tissues

• Blood vessels bring oxygen and nutrients via the circulation to nourish all tissues in the body

• The cells comprising blood vessels are called endothelial cells

• The endothelial cells of a blood vessel also produce molecules that support the growth of tissues

• Cancer cells take over the body's control of angiogenesis in order to recruit their own private blood supply

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Historical Highlights of the

Anti-Angiogenesis Field

Historical Highlights of the

Anti-Angiogenesis Field • 1787 - British surgeon Dr. John Hunter first uses the term 'angiogenesis' (new blood vessel growth) to

describe blood vessels growing in the reindeer antler

• 1971 - Surgeon Dr. Judah Folkman hypothesizes that tumor growth is dependent upon angiogenesis. His theory, published in the New England Journal of Medicine, and is initially regarded as heresy by leading physician and scientists.

• 1975 - The first angiogenesis inhibitor is discovered in cartilage by Dr. Henry Brem and Dr. Judah Folkman.

• 1984 - The first angiogenic factor (basic fibroblast growth factor, bFGF) is purified by Yuen Shing and Michael Klagsbrun at Harvard Medical School.

• 1989 - One of the most important angiogenic factors, vascular endothelial growth factor (VEGF), is

discovered by Dr. Napoleone Ferrara and by Dr. Jean Plouet. It turns out to be identical to a molecule called Vascular Permeability Factor (VPF) discovered in 1983 by Dr. Harold Dvorak.

• 1787 - British surgeon Dr. John Hunter first uses the term 'angiogenesis' (new blood vessel growth) to

describe blood vessels growing in the reindeer antler

• 1971 - Surgeon Dr. Judah Folkman hypothesizes that tumor growth is dependent upon angiogenesis. His theory, published in the New England Journal of Medicine, and is initially regarded as heresy by leading physician and scientists.

• 1975 - The first angiogenesis inhibitor is discovered in cartilage by Dr. Henry Brem and Dr. Judah Folkman.

• 1984 - The first angiogenic factor (basic fibroblast growth factor, bFGF) is purified by Yuen Shing and Michael Klagsbrun at Harvard Medical School.

• 1989 - One of the most important angiogenic factors, vascular endothelial growth factor (VEGF), is

discovered by Dr. Napoleone Ferrara and by Dr. Jean Plouet. It turns out to be identical to a molecule called Vascular Permeability Factor (VPF) discovered in 1983 by Dr. Harold Dvorak.

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Historical Highlights of the

Anti-Angiogenesis Field

Historical Highlights of the

Anti-Angiogenesis Field• 1997 - Dr. Michael O'Reilly publishes research finding in the journal Nature showing complete regression of cancerous tumors following repeated cycles of anti-angiogenic therapy

using angiostatin and endostatin • 1999 - Massive wave of anti-angiogenic drugs in

clinical trials: 46 anti- angiogenic drugs for cancer patients; 5 drugs for macular degeneration; 1 drug for diabetic retinopathy; 4 drugs

for psoriasis. • 1999 - Dr. Richard Klausner, Director of the U.S.

National Cancer Institute designates the development of anti-angiogenic therapies for cancer as a national priority.

• 2003 - The monoclonal antibody drug Avastin (Bevacizumab) becomes the first anti-angiogenic drug shown in large-scale clinical trials inhibiting tumor blood vessel growth can prolong survival in cancer patients.

• 1997 - Dr. Michael O'Reilly publishes research finding in the journal Nature showing complete regression of cancerous tumors following repeated cycles of anti-angiogenic therapy

using angiostatin and endostatin • 1999 - Massive wave of anti-angiogenic drugs in

clinical trials: 46 anti- angiogenic drugs for cancer patients; 5 drugs for macular degeneration; 1 drug for diabetic retinopathy; 4 drugs

for psoriasis. • 1999 - Dr. Richard Klausner, Director of the U.S.

National Cancer Institute designates the development of anti-angiogenic therapies for cancer as a national priority.

• 2003 - The monoclonal antibody drug Avastin (Bevacizumab) becomes the first anti-angiogenic drug shown in large-scale clinical trials inhibiting tumor blood vessel growth can prolong survival in cancer patients.

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Specific Angiogenic Inhibitors

Specific Angiogenic Inhibitors

•Angiostatin•Avastin (Bevacizumab)•Celebrex (Celecoxib)•Endostatin•Metaret (Suramin)•Thalidomide

•Angiostatin•Avastin (Bevacizumab)•Celebrex (Celecoxib)•Endostatin•Metaret (Suramin)•Thalidomide

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AngiostatinAngiostatin• Naturally occurring

protein found in several animal species, including humans.

• It is an endogenous angiogenesis inhibitor

• Angiostatin is produced by autoproteolytic cleavage of plasminogen,

• Can be cleaved from plasminogen by different metalloproteinases (MMPs), elastase, prostata-specific antigen (PSA), 13 KD serine protease, or 24KD endopeptidase.

• Naturally occurring protein found in several animal species, including humans.

• It is an endogenous angiogenesis inhibitor

• Angiostatin is produced by autoproteolytic cleavage of plasminogen,

• Can be cleaved from plasminogen by different metalloproteinases (MMPs), elastase, prostata-specific antigen (PSA), 13 KD serine protease, or 24KD endopeptidase.

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AngiostatinAngiostatin

• It is a 57 kDa fragment of a larger protein, Plasmin (itself a fragment of plasminogen)

• Encloses three to five contiguous Kringle modules.

• Each Kringle module contains two small beta sheets and three disulfide bonds.

• Considerable uncertainty on its mechanism of action, but it seems to involve the inhibition of endothelial cell migration, proliferation and induction of apoptosis.

• It is a 57 kDa fragment of a larger protein, Plasmin (itself a fragment of plasminogen)

• Encloses three to five contiguous Kringle modules.

• Each Kringle module contains two small beta sheets and three disulfide bonds.

• Considerable uncertainty on its mechanism of action, but it seems to involve the inhibition of endothelial cell migration, proliferation and induction of apoptosis.

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AvastinAvastin• Avastin is a humanized monoclonal antibody (MAb) that targets vascular endothelial growth factor (VEGF)

• Causes regression of tumor vasculature • Reduces intra-tumor pressure, thereby improving the delivery of cytotoxic agents to the tumor

• Also inhibits new tumor blood vessel formation, restricting tumor growth.

• The first anti-angiogenic agent with demonstrated anticancer benefit in phase III trials.

• Avastin-VEGF Animation

• Avastin is a humanized monoclonal antibody (MAb) that targets vascular endothelial growth factor (VEGF)

• Causes regression of tumor vasculature • Reduces intra-tumor pressure, thereby improving the delivery of cytotoxic agents to the tumor

• Also inhibits new tumor blood vessel formation, restricting tumor growth.

• The first anti-angiogenic agent with demonstrated anticancer benefit in phase III trials.

• Avastin-VEGF Animation

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Celecoxib Celecoxib • Is one of the “rediscovered” drugs• It is better known as Celebrex, a non-steroidal, anti-

inflammatory drug• Celecoxib is a COX-2 inhibitor

– Overexpression of COX-2 in cancer cells induces the production of VEGF, PDGF, bFGF and TGF-beta.

– Through these angiogenesis mediators and their receptors on the endothelial cells, COX-2 increased vascular permeability and induced endothelial cell proliferation and migration.

– COX-2 overexpression led to the production of matrix metalloproteinase (MMPs), which have been implicated in extracellular matrix invasion

– COX enzymes are essential for maintenance of the migration and attachment of endothelial cells through integrin pathways

• Therefore a COX-2 inhibitor will block new vessel formation

• Is one of the “rediscovered” drugs• It is better known as Celebrex, a non-steroidal, anti-

inflammatory drug• Celecoxib is a COX-2 inhibitor

– Overexpression of COX-2 in cancer cells induces the production of VEGF, PDGF, bFGF and TGF-beta.

– Through these angiogenesis mediators and their receptors on the endothelial cells, COX-2 increased vascular permeability and induced endothelial cell proliferation and migration.

– COX-2 overexpression led to the production of matrix metalloproteinase (MMPs), which have been implicated in extracellular matrix invasion

– COX enzymes are essential for maintenance of the migration and attachment of endothelial cells through integrin pathways

• Therefore a COX-2 inhibitor will block new vessel formation

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CelecoxibCelecoxib

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EndostatinEndostatin• It was first

discovered in 1995 in Dr. Folkman’s lab

• Phase I clinical studies began at M.D. Anderson November 1999

• A naturally-occurring 20-kDa C-terminal fragment derived from type XVIII collagen.

• Interfere with the pro-angiogenic action of growth factors such as basic fibroblast growth factor (bFGF/FGF-2) and vascular endothelial growth factor (VEGF)

• It was first discovered in 1995 in Dr. Folkman’s lab

• Phase I clinical studies began at M.D. Anderson November 1999

• A naturally-occurring 20-kDa C-terminal fragment derived from type XVIII collagen.

• Interfere with the pro-angiogenic action of growth factors such as basic fibroblast growth factor (bFGF/FGF-2) and vascular endothelial growth factor (VEGF)

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SuraminSuramin

• Developed by Oskar Dressel and Richard Kothe of Bayer, Germany in 1916

• A polysulfonated naphthylurea• Is a prototype of a pharmacological antagonist of growth factors,

including basic fibroblast growth factor (bFGF)• It is usually used for treatment of human sleeping sickness,

onchocerciasis and other diseases caused by trypanosomes and worms

• Developed by Oskar Dressel and Richard Kothe of Bayer, Germany in 1916

• A polysulfonated naphthylurea• Is a prototype of a pharmacological antagonist of growth factors,

including basic fibroblast growth factor (bFGF)• It is usually used for treatment of human sleeping sickness,

onchocerciasis and other diseases caused by trypanosomes and worms

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ThalidomideThalidomide• One of the most perplexing drugs in medical history.

• It is a hypnotic, causes peripheral nerve damage and severe birth defects, is anti-inflammatory, enhances the immune system, inhibits HIV replication, and inhibits some cancers

• Thalidomide decreases TNF-a, tumor necrosis factor alpha levels, – TNF causes apoptotic cell death, – cellular proliferation, – differentiation, – inflammation, and – tumorigenesis

• One of the most perplexing drugs in medical history.

• It is a hypnotic, causes peripheral nerve damage and severe birth defects, is anti-inflammatory, enhances the immune system, inhibits HIV replication, and inhibits some cancers

• Thalidomide decreases TNF-a, tumor necrosis factor alpha levels, – TNF causes apoptotic cell death, – cellular proliferation, – differentiation, – inflammation, and – tumorigenesis

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ThalidomideThalidomide

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ThalidomideThalidomide

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SummarySummary• Angiogenesis inhibitors specifically halt new blood

vessel growth and starve a tumor by cutting off its blood supply.

• VEGF is responsible for the growth of new blood vessels. It promotes this growth by stimulating the endothelial cells, which form the walls of the vessels and transport nutrients and oxygen to the tissues.

• Angiogenesis inhibitors prevent the VEGF from binding with the receptors on the surface of the endothelial cells.

• There are 3 major types of anti-angiogenic therapies• Angiogenesis is the growth of blood vessels and is an

important natural process used by the body for reproduction and for healing injured tissues

• Angiogenesis inhibitors specifically halt new blood vessel growth and starve a tumor by cutting off its blood supply.

• VEGF is responsible for the growth of new blood vessels. It promotes this growth by stimulating the endothelial cells, which form the walls of the vessels and transport nutrients and oxygen to the tissues.

• Angiogenesis inhibitors prevent the VEGF from binding with the receptors on the surface of the endothelial cells.

• There are 3 major types of anti-angiogenic therapies• Angiogenesis is the growth of blood vessels and is an

important natural process used by the body for reproduction and for healing injured tissues

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CitationsCitations1. O’Reilly, M.S., et al., Cell, 79, 315 (1994). 

Folkman, J., Nat. Med., 1, 27 (1995). Sim, S.K., et al., Cancer Res., 57, 1329 (1997). 

2. Wu, Z., et al., Biochem. Biophys. Res. Commun., 236, 651 (1997). 3. Boehm, T. Folkman, J. Browder, T. & M. O’Reilly : Anti-angiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature

390, 404-407 (1997) 4. Kim, K.J. Li, B. Winer, J. Armanini, M. Gillett, N. Phillips, H.S. & N. Ferrara : Inhibition of vascular endothelial growth factor-induced

angiogenesis suppresses ttumor growth in vivo. Nature 362, 841-844 (1993) 5. Robinson, G.S. Pierce, E.A. Rook, S.L. Foley, E. Webb, R. & L.E. Smith: Oligodeoxynucleotides inhibit retinal neovascularization in a murine model

of proliferative retinopathy. Proc Natl Acad Sci USA 93, 4851-4856 (1996) 6. Aiello, L.P. Pierce, E.A. Foley, E.D. Takagi, H. Chen, H. Riddle, L. Ferrara, N. King, G.L. & L.E. Smith: Suppression of retinal

neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci USA 92, 10457-10461 (1995)

7. Goldman, c., Kendall, R., Cabrera, G., Soroceanu, L., Heike, Y., Gillespie, G., Siegal, G., Mao, X., Bett, A., Huckle, W., Thomas, K. & D. Curiel: Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate Proc. Natl. Acad. Sci.USA 95, 8875-8800 (1998)

8. Skobe, M., Rockwell, P., Goldstein, N., Vosseler, S., Fusening, N.: Halting angiogenesis suppresses carcinoma cell invasion Nature Med 3, 1222-1227 (1997)

9. Warren, R.S., Yuan H., Matli M.R., Gillett N.A. & N. Ferrara: Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis. J Clin Invest 1995; 95: 1789-1797.

10. Claesson-Welsh, L., Welsh, M., Ito, N., Anand-Apte, B., Soker, S., Zetter, B., O'Reilly, M. & J. Folkman: Angiogastin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD . Proc Natl Acad Sci USA 95, 5579-5583 (1998)

11. Clauss, M. Weich, H. Breier, G. Knies, U. Rockl, W. Waltenberger, J. & W. Risau: The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis. J Biol Chem 271, 17629-17634 (1996)

12. Jonca, F. Ortéga, N. Gleizes, P.E. Bertrand, N. & J. Plouët: Cell release of bioactive fibroblast growth factor by exon 6 encoded sequence of vascular endothelial growth factor. J Biol Chem 272, 24203-24209 (1997)

13. Waltenberger, J. Claesson-Welsh, L. Siegbahn, A. Shibuya, M. & C.H. Heldin: Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J Biol Chem 269, 26988-26995 (1994)

14. Joukov, V. Sorsa, T. Kumar, V. Jeltsch, M. Claesson-Welsh, L. Cao, Y. Saksela, O. Kalkkinen, N. & K. Alitalo: Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J 13, 3898-3911 (1997)

15. M.P. Seed, J.R. Brown, C.N. Freemantle, J.L. Papworth, P.R. Colville-Nash, D. Willis, K.W. Somerville, S. Asculai, D.A. Willoughby, The inhibition of colon-26 adenocarcinoma development and angiogenesis by topical diclofenac in 2.5% hyaluronan, Cancer Res. 57 (1997) 1625-1629.

16. T.O. Daniel, H. Liu, J.D. Morrow, B.C. Crews, L.J. Marnett, Thromboxane A2 is a mediator of cyclooxygenase-2-dependent endothelial migration and angiogenesis, Cancer Res. 59 (1999) 4574-4577.

17. E. Fosslien, Review: molecular pathology of cyclooxygenase-2 in cancer-induced angiogenesis, Ann. Clin. Lab Sci. 31 (2001) 325-348. 18. S. Hockel, K. Schlenger, P. Vaupel, M. Hockel, Association between host tissue vascularity and the prognostically relevant tumor vascularity in

human cervical cancer, Int. J. Oncol. 19 (2001) 827-832. 19. D.W. Visscher, S. Smilanetz, S. Drozdowicz, S.M. Wykes, Prognostic significance of image morphometric microvessel enumeration in breast carcinoma,

Anal. Quant. Cytol. Histol. 15 (1993) 88-92. 20. M. Tsujii, S. Kawano, S. Tsuji, H. Sawaoka, M. Hori, R.N. DuBois, Cyclooxygenase regulates angiogenesis induced by colon cancer cells [published

erratum appears in Cell 1998 Jul 24;94(2):following 271], Cell 93 (1998) 705-716. 21. P. Pradono, R. Tazawa, M. Maemondo, M. Tanaka, K. Usui, Y. Saijo, K. Hagiwara, T. Nukiwa, Gene transfer of thromboxane A(2) synthase and

prostaglandin I(2) synthase antithetically altered tumor angiogenesis and tumor growth, Cancer Res. 62 (2002) 63-66. 22. Y. Takahashi, F. Kawahara, M. Noguchi, K. Miwa, H. Sato, M. Seiki, H. Inoue, T. Tanabe, T. Yoshimoto, Activation of matrix metalloproteinase-2 in

human breast cancer cells overexpressing cyclooxygenase-1 or -2, FEBS Lett. 460 (1999) 145-148.

1. O’Reilly, M.S., et al., Cell, 79, 315 (1994). Folkman, J., Nat. Med., 1, 27 (1995). Sim, S.K., et al., Cancer Res., 57, 1329 (1997). 

2. Wu, Z., et al., Biochem. Biophys. Res. Commun., 236, 651 (1997). 3. Boehm, T. Folkman, J. Browder, T. & M. O’Reilly : Anti-angiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature

390, 404-407 (1997) 4. Kim, K.J. Li, B. Winer, J. Armanini, M. Gillett, N. Phillips, H.S. & N. Ferrara : Inhibition of vascular endothelial growth factor-induced

angiogenesis suppresses ttumor growth in vivo. Nature 362, 841-844 (1993) 5. Robinson, G.S. Pierce, E.A. Rook, S.L. Foley, E. Webb, R. & L.E. Smith: Oligodeoxynucleotides inhibit retinal neovascularization in a murine model

of proliferative retinopathy. Proc Natl Acad Sci USA 93, 4851-4856 (1996) 6. Aiello, L.P. Pierce, E.A. Foley, E.D. Takagi, H. Chen, H. Riddle, L. Ferrara, N. King, G.L. & L.E. Smith: Suppression of retinal

neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci USA 92, 10457-10461 (1995)

7. Goldman, c., Kendall, R., Cabrera, G., Soroceanu, L., Heike, Y., Gillespie, G., Siegal, G., Mao, X., Bett, A., Huckle, W., Thomas, K. & D. Curiel: Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate Proc. Natl. Acad. Sci.USA 95, 8875-8800 (1998)

8. Skobe, M., Rockwell, P., Goldstein, N., Vosseler, S., Fusening, N.: Halting angiogenesis suppresses carcinoma cell invasion Nature Med 3, 1222-1227 (1997)

9. Warren, R.S., Yuan H., Matli M.R., Gillett N.A. & N. Ferrara: Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis. J Clin Invest 1995; 95: 1789-1797.

10. Claesson-Welsh, L., Welsh, M., Ito, N., Anand-Apte, B., Soker, S., Zetter, B., O'Reilly, M. & J. Folkman: Angiogastin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD . Proc Natl Acad Sci USA 95, 5579-5583 (1998)

11. Clauss, M. Weich, H. Breier, G. Knies, U. Rockl, W. Waltenberger, J. & W. Risau: The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis. J Biol Chem 271, 17629-17634 (1996)

12. Jonca, F. Ortéga, N. Gleizes, P.E. Bertrand, N. & J. Plouët: Cell release of bioactive fibroblast growth factor by exon 6 encoded sequence of vascular endothelial growth factor. J Biol Chem 272, 24203-24209 (1997)

13. Waltenberger, J. Claesson-Welsh, L. Siegbahn, A. Shibuya, M. & C.H. Heldin: Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J Biol Chem 269, 26988-26995 (1994)

14. Joukov, V. Sorsa, T. Kumar, V. Jeltsch, M. Claesson-Welsh, L. Cao, Y. Saksela, O. Kalkkinen, N. & K. Alitalo: Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J 13, 3898-3911 (1997)

15. M.P. Seed, J.R. Brown, C.N. Freemantle, J.L. Papworth, P.R. Colville-Nash, D. Willis, K.W. Somerville, S. Asculai, D.A. Willoughby, The inhibition of colon-26 adenocarcinoma development and angiogenesis by topical diclofenac in 2.5% hyaluronan, Cancer Res. 57 (1997) 1625-1629.

16. T.O. Daniel, H. Liu, J.D. Morrow, B.C. Crews, L.J. Marnett, Thromboxane A2 is a mediator of cyclooxygenase-2-dependent endothelial migration and angiogenesis, Cancer Res. 59 (1999) 4574-4577.

17. E. Fosslien, Review: molecular pathology of cyclooxygenase-2 in cancer-induced angiogenesis, Ann. Clin. Lab Sci. 31 (2001) 325-348. 18. S. Hockel, K. Schlenger, P. Vaupel, M. Hockel, Association between host tissue vascularity and the prognostically relevant tumor vascularity in

human cervical cancer, Int. J. Oncol. 19 (2001) 827-832. 19. D.W. Visscher, S. Smilanetz, S. Drozdowicz, S.M. Wykes, Prognostic significance of image morphometric microvessel enumeration in breast carcinoma,

Anal. Quant. Cytol. Histol. 15 (1993) 88-92. 20. M. Tsujii, S. Kawano, S. Tsuji, H. Sawaoka, M. Hori, R.N. DuBois, Cyclooxygenase regulates angiogenesis induced by colon cancer cells [published

erratum appears in Cell 1998 Jul 24;94(2):following 271], Cell 93 (1998) 705-716. 21. P. Pradono, R. Tazawa, M. Maemondo, M. Tanaka, K. Usui, Y. Saijo, K. Hagiwara, T. Nukiwa, Gene transfer of thromboxane A(2) synthase and

prostaglandin I(2) synthase antithetically altered tumor angiogenesis and tumor growth, Cancer Res. 62 (2002) 63-66. 22. Y. Takahashi, F. Kawahara, M. Noguchi, K. Miwa, H. Sato, M. Seiki, H. Inoue, T. Tanabe, T. Yoshimoto, Activation of matrix metalloproteinase-2 in

human breast cancer cells overexpressing cyclooxygenase-1 or -2, FEBS Lett. 460 (1999) 145-148.